The progression of the asymptomatic hemisphere of moyamoya disease (MMD) is largely unknown. In this study, we investigated the differences in subcortical gray matter structure and angiographic features between asymptomatic and symptomatic hemispheres in patients with MMD.We retrospectively reviewed patients with MMD in consecutive cases in our center. We compared subcortical gray matter volume and three types of collaterals  between symptomatic and asymptomatic hemispheres. Symptomatic hemispheres were classified as ischemic hemisphere (i-hemisphere) and hemorrhagic hemisphere (h-hemisphere). Asymptomatic hemispheres were classified as contralateral asymptomatic hemisphere of i-hemisphere (ai-hemisphere), contralateral asymptomatic hemisphere of h-hemisphere (ah-hemisphere), bilateral asymptomatic hemispheres in asymptomatic group (aa-hemisphere).P = 0.010), between the i- and ah-hemispheres (P = 0.004), as well as between the h- and ai-hemispheres (P = 0.002), between the h- and ah-hemispheres (P < 0.001). There was a higher incidence of thalamic anastomosis in the ai-hemispheres than i-hemispheres , and in the ah-hemispheres than h-hemispheres . Additionally, the hemispheres with thalamic anastomosis had a significantly greater volume than those without thalamic anastomosis (P = 0.024). Univariate and multivariate logistic regression analysis showed that thalamic volume was closely associated with thalamic anastomosis.A total of 117 MMD patients were reviewed, and 49 of them met the inclusion criteria, with 98 hemispheres being analyzed. The thalamic volume was found to differ significantly between the i- and ai-hemispheres (The thalamic volume and the incidence of thalamic anastomosis increase in asymptomatic hemispheres and decrease in symptomatic hemispheres. Combining these two characteristics may be helpful in assessing the risk of stroke in the asymptomatic hemispheres of MMD as well as understanding the pathological evolution of the disease. Moyamoya disease (MMD) is a type of chronic cerebrovascular disease characterized by progressive steno-occlusive changes at the terminal of the internal carotid arteries and the puff of smoke-like vascular network at the base of the brain . Cerebra“Moyamoya” vessels are the notably dilated perforating arteries in the basal ganglia and thalamus that conduct an important collateral circulation . AbnormaThe aim of this study was to explore the differences of subcortical gray matter structure and angiographic features between asymptomatic and symptomatic hemispheres in patients with MMD. Clarifying the structural and angiographic characteristics of the asymptomatic hemisphere would be helpful to further understand the natural course of MMD and guide management of the asymptomatic hemispheres.We retrospectively reviewed consecutive patients diagnosed with MMD who visited our institution between July 2020 and May 2022. The diagnostic criteria were based on digital subtraction angiography (DSA) or magnetic resonance angiography (MRA) which were in accordance with the guideline . We enroThe exclusion criteria were as follows: (1) imaging data did not meet the imaging protocols or angiography lost; (2) hemorrhagic MMD attributed to ruptured intracranial aneurysms or which side of the brain suffering from hemorrhage could not be distinguished; (3) infarction or lesion in subcortical gray matter was excluded, which was considered to affect the structure of the subcortical region. The patients’ inclusion and exclusion flowchart was displayed in According to the clinical presentations and radiological findings, the included MMD patients were divided into ischemic group, hemorrhagic group, and asymptomatic group . Their hemispheres were classified into five types proposed by The MR imaging was completed on a 3T MR scanner  with an 8-channel head coil. T1-weighted images (T1WI) were collected through 3D fast spoiled gradient-echo with repetition time (TR), 7.4 ms; echo time (TE), 3.1 ms; flip angle (FA), 8°; field of view (FOV), 256 mm × 256 mm; slices, 170; voxel size, 0.8 mm × 0.8 mm × 1.0 mm. T2-Flair images were acquired with TR, 8,400 ms; TE, 145.3 ms; FA, 90°; FOV, 512 mm × 512 mm; slices, 40; voxel size, 0.39 mm × 0.39 mm × 4.0 mm. Though it takes more scanning time for T1WI with high resolution, we get a more accurate structure of the subcortical gray matter. Time-of-flight MRA images were obtained by TR, 21 ms; TE, 2.5 ms; FA, 20°; FOV, 512 mm × 512 mm; slices, 129; voxel size, 0.39 mm × 0.39 mm × 1.4 mm.v6.01 with the “recon-all” pipeline to automatically perform 31 processing steps for each subject, the technical details of these procedures were described in prior publications  who were completely blinded to the patients’ clinical information through the anteroposterior and lateral views on DSA. If there was any difference, another senior neurosurgeon (Dr. Wang) was invited to discuss and reach a final consensus.t-tests and Mann–Whitney U tests were used as appropriate when performed to determine group difference. The difference in the subcortical volume between groups was compared using the one-way ANOVA tests. The proportion of positive anastomosis and other categorical variables between groups were compared by the chi-square test. All variables from the univariate analysis using binary logistic regression with a P < 0.15 were further moved into a forward-stepwise multivariate analysis. Analyses were performed using SPSS v25 software  and GraphPad Prism v8.0 . Two-sided values of P < 0.05 was considered as statistically significant. All data generated and analyzed are available on reasonable request.Continuous variables were presented as the mean ± SD. The In this consecutive cohort of 117 patients with MMD, 10 patients had no documented DSA data; 18 patients had significant lesion in subcortical gray matter region; five patients had hemorrhage due to ruptured aneurysm; In three patients it was unable to distinguish the side of hemorrhage because of the equal volume of intraventricular hemorrhage in both sides; 31 patients did not met the imaging protocols of high-resolution T1WI; and 1 patient had an unknown error when performing image processing. The aforementioned patients were excluded from this study.n = 7, 43.8%), cerebral ischemia , and paresthesia . Twenty-seven patients were hemorrhagic MMD (including both 27 h- and ah-hemispheres) with the most frequent manifestation was primary intraventricular hemorrhage (IVH) , followed by IVH with intracerebral hemorrhage (ICH) , subarachnoid hemorrhage (SAH) , IVH with SAH , and ICH only . And 6 cases in the asymptomatic group with 12 aa-hemispheres.Finally, 49 patients with 98 hemispheres were enrolled. Among them, there were 16 patients in the ischemic group (including both 16 i- and ai-hemispheres) with symptoms including transient ischemic attack , cerebral ischemia , dizziness , and headache . A total of 10 patients were hemorrhagic MMD with the most frequent manifestation was primary IVH , followed by IVH with ICH , IVH with subarachnoid hemorrhage , and ICH only .A total of 12 patients were ischemic MMD with symptoms including transient ischemic attack (P = 0.006).P = 0.010), the i- and ah-hemispheres (P = 0.004), the h- and ai-hemispheres (P = 0.002), as well as the h- and ah-hemispheres (P < 0.001) (P = 0.030), and the i- and ah-hemispheres (P = 0.034)  . The put= 0.034) . As for = 0.034) . The tot= 0.034) .P = 0.070), and between the h- and ah-hemispheres  (P = 0.032)  . The h-h= 0.032) . Table 2P = 0.024) (P = 0.105), BMI  and thalamic volume  were associated with thalamic anastomosis. Furthermore, the multivariate logistic analysis indicated that thalamic volume  was the only factor that related to thalamic anastomosis  . In addistomosis .Identifying the brain volume, which is an important component of brain structure , as wellCerebral blood supply and hemodynamics are closely related to the brain structure , decreasWe next investigated the angiographic feature between the hemispheres. The proportion of three different types of collateral anastomosis in this cohort was comparable to that of previous studies . There wIncreased thalamic volume and the incidence of thalamic anastomosis in the asymptomatic hemisphere prompted us to explore the relationship between the two characteristics. Thalamic anastomosis arises from the thalamotuberal artery and thalamoperforating artery, which originate from the posterior communicating artery and posterior cerebral artery, respectively . Both ofThe results of this study can be interpreted in two different ways. The first is that the hemispheres with a small thalamic volume and a lower incidence of thalamic anastomosis are more likely to suffer a stroke. The second assumption is that in the symptomatic hemispheres, thalamic anastomosis contracts after a stroke attack, which further causes atrophy of the thalamus. While in the ai- and ah-hemispheres, the thalamic volume and thalamic anastomosis are better preserved. Therefore, the combination of these two characteristics may provide insight into the risk of stroke in the asymptomatic hemispheres of MMD and the pathological development of the disease.There are several limitations to be noted. Firstly, after taking into account certain factors that may influence the results of this study, the sample size appears to be small, which, in turn, limits the generalizability of the findings. A multicenter, large-sample study is needed to verify our results. Secondly, all of the patients included in this study were independent in their daily lives (scores between 0 and 2 on mRS), which might lead to a selection bias. Thirdly, In adult patients with MMD, the thalamic volume and the incidence of thalamic anastomosis increase in asymptomatic hemispheres and decrease in symptomatic hemispheres. Combining these two characteristics may be helpful in assessing the risk of stroke in the asymptomatic hemispheres of MMD as well as understanding the pathological evolution of the disease.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Human Research Ethics Committee of the Second Affiliated Hospital of Zhejiang University (ID: 2020-064). The patients/participants provided their written informed consent to participate in this study.JH and YW wrote the manuscript and collected the data. YT revised the manuscript. GL helped to evaluate statistical results. YL and JC assisted in writing the manuscript and contributed to the analysis of data. DX assisted in the MRI scan. JH, YL, and LW reviewed all imaging data. LW and RB conceived and designed the manuscript. All authors contributed to this article and approved the submitted version.


Background and Objectives: A significant role was played by costimulatory molecules in renal cancer. However, the lncRNAs regulating costimulatory molecules have not been fully investigated. Materials and Methods: Data from the next-sequence file and clinical data were downloaded from the Cancer Genome Atlas (TCGA) database. All analyses were conducted using the R and GraphPad Prism software. Results: A total of 1736 costimulatory molecule-related lncRNAs were determined under the threshold of |Cor| > 0.5 and p-value < 0.001. Furthermore, a prognosis prediction signature consisting of five lncRNAs: LINC00941, AC016773.1, AL162171.1, HOTAIRM1, and AL109741.1 was established with great prediction ability. By combining risk score and clinical parameters, a nomogram plot was constructed for better clinical practice. A biological enrichment analysis indicated that E2F targets, coagulation, IL6/JAK/STAT3 signaling, G2/M checkpoint, and allograft rejection pathways were activated in high-risk patients. Furthermore, a higher infiltration level of resting CD4+ T cell, M2 macrophage, and resting mast cells, while a lower CD8+ T cell infiltration was observed in high-risk patients. It is worthy of note that, low-risk patients might respond better to PD-1 checkpoint therapy. A correlation analysis of LINC00941 revealed that it was positively correlated with Th2 cells, Th1 cells, macrophages, and Treg cells, but negatively correlated with Th17 cells. A pathway enrichment analysis indicated that the pathways of the inflammatory response, G2M checkpoint, and IL6/JAK/STAT3 signaling were significantly activated in patients with high LINC00941 expression. In vitro experiments indicated that LINC00941 can enhance the malignant biological behaviors of renal cancer cells. Conclusions: Our study established a costimulatory molecule-related lncRNAs-based prognosis model with a great prediction prognosis. In addition, LINC00941 could enhance the malignant biological behaviors of renal cancer cells. In 2020, there were approximately 420,000 new cases of renal cell carcinoma (RCC) and 180,000 deaths from it globally . MoreoveCostimulatory molecules, consisting of the B7-CD28 family and tumor necrosis factor (TNF) family, play a critical role in cancer biology . Cell-meIncreasing amounts of public data have been generated in the era of Big Data, and secondary analysis of these data can provide researchers with direction . Here, wData from the next-sequence file and clinical data were downloaded from the Cancer Genome Atlas (TCGA) database of the TCGA-KIRC project . The original form of expression profile data was the “STAR-count” form. All the data were preprocessed before analysis. The detailed clinicopathological parameters of patients enrolled in our analysis are shown in p-value < 0.001.A correlation analysis was utilized to identify the relationship between costimulatory molecules and lncRNAs data. The lncRNAs were considered as CMLs meeting the criteria of |Cor| > 0.5 and p value < 0.05). Following this, the Random Survival Forests Variable Hunting (RSFVH) algorithm was utilized to identify lncRNAs. Additionally, independent prognostic factors associated with CML survival were determined by a multivariate Cox regression analysis. The formula of the risk score consisting of five CMLs was as follows:For the enrolled patients, a 1:1 ratio was used to randomly assign patients to training and validation groups. First, using a Univariate Cox regression analysis, CMLs associated with patient survival were identified.  survival curve. In the training and validation cohorts, the median value of the risk score in each cohort was defined as the cut-off values to distinguish high- and low-risk groups. The patients with a risk score higher than the median value were high-risk patients, otherwise, they were considered to be low-risk patients.Using the rms package, a nomogram was established for better application in the clinic. Furthermore, the calibration curve for 3-, 5-, and 8 years was performed to compare the predictive accuracies of the nomogram.n = 9) retrieved from the MSigDB database were chosen as the gene set of reference.To investigate the significant differences between the two risk groups, a gene set enrichment analysis (GSEA) was performed. All annotated gene set files . The method for p value correction was “Bonferroni” and the output Bonferroni corrected p value could reflect the similarity between selected patients and specific immunotherapy therapy cohorts , in which Bonferroni corrected p value < 0.05 was considered as statistically significant.The proportions of 22 different types of infiltrating immune cells in the tumor microenvironment were quantified using the CIBERSORT algorithm . Each paNormal renal epithelial cell lines (HK-2) and renal cancer cell lines  were purchased from the Cell Bank of Shanghai Academy of Chinese Sciences and routinely maintained at our laboratory. Total RNA was extracted using an RNA extraction kit (Qiagen). The sequences of primers used in the study were as follows: LINC00941: forward primer, 5′-CAAGCAACCGTCCAACTACCAGACA-3′; reverse primer, 5′-AAATCAAGAGCCCAAACATTGTGAA-3′; GAPDH, forward primer, 5′-CTGGGCTACACTGAGCACC-3′, forward primer, 5′-AAGTGGTCGTTGAGGGCAATG-3′.Cell transfection was carried out based on the lipofectamine 3000 regent following the protocol. The control and sh-LINC00941 plasmids were purchased from Shanghai GenePharma, whose target sequences were as follows: sh-LINC#1, 5′-GGACCAACTATGCTTATAA-3′; sh-LINC#2, 5′-GCCCTCGAGAAGTGTCTAA-3′; sh-LINC#3, 5′-GAGCATGTATCCATCTTAT-3′.Firstly, we resuspended and seeded 500 cells per well in a six-plate well. The cells were then cultured for 14 days according to conventional cell culture conditions. Finally, crystal violet staining was applied after the cells were mixed with formaldehyde. A CCK8 assay was conducted using a CCK8 kit  based on the protocol.3 cells with medium FBS. The lower chamber was filled with a medium containing 20% FBS. Cells were then stained with crystal violet after being mixed with formaldehyde for 24 h.In a 24-plate well, an 8-um pore Transwell chamber was used to divide the plate into the upper and lower chamber. To upper chamber were added 4 × 10p-value less than 0.05. For the variables conforming to a normal distribution, a Student’s t-test was used for analysis; for the variables conforming to non-normal distribution, the Mann-Whitney U test was used for analysis. All biological experiments were repeated three times to obtain a statistical p value. The receiver operating characteristic (ROC) curve was utilized to assess the prediction performance of the identified variables. The Area Under the Curve (AUC) value of the ROC curve was calculated using the survivalROC package in R software.All statistical analyses were conducted using R software and GraphPad Prism 8. Statistical significance was determined by a two-sided p value = 0.001 , affecting the development of various cancer including ccRCC . Our stuOn the other hand, a pathway enrichment analysis indicated that the G2/M checkpoints and E2F targets were activated in high-risk groups. In the cell cycle, the G2/M checkpoint is crucial. The abnormal state of the G2/M checkpoint could lead to unrestricted proliferation ability . Most tuBased on our analysis, LINC00941 had the highest importance in identified CMLs and was therefore selected for further research. LINC00941 has been reported in multiple cancers. For example, in colon cancer, Wu et al. indicated that by preventing the degradation of SMAD4 protein, LINC00941 facilitates cancer metastasis . In papiIn this study, we successfully constructed a CMLs-based prognosis model and demonstrated the oncogenic role of LINC00941 in ccRCC. However, it invariably has some limitations that need to be addressed. Firstly, the patients enrolled in the analysis were predominantly Caucasian, which introduced a bias to the application of our conclusion to other populations. Secondly, due to the limitations of data, we only performed an internal validation for our survival prediction model based on TCGA data. Thirdly, the TCGA database has no laboratory findings of enrolled patients, which might bring potential bias to the evaluation of the overall condition of the patients. Fourthly, the underlying mechanism of LINC00941 in ccRCC has not been fully explored.


Recently approved mRNA vaccines are administered intramuscularly where they transfect antigen-presenting cells (APCs) near the site of administration, resulting in an immune response. The spleen contains high numbers of APCs, which are located near B and T lymphocytes. Therefore, transfecting APCs in the spleen would be expected to produce a more efficient immune response, but this is a challenging task due to the different biological barriers. Success requires the development of an efficient system that can transfect different immune cells in the spleen. In this study, we report on the development of mRNA-loaded lipid nanoparticles (LNPs) targeting immune cells in the spleen with the goal of eliciting an efficient immune response against the antigen encoded in the mRNA. The developed system is composed of mRNA loaded in LNPs whose lipid composition was optimized for maximum transfection into spleen cells. Dendritic cells, macrophages and B cells in the spleen were efficiently transfected. The optimized LNPs produced efficient dose-dependent cytotoxic T lymphocyte activities that were significantly higher than that produced after local administration. The optimized LNPs encapsulating tumor-antigen encoding mRNA showed both prophylactic and therapeutic antitumor effects in mice. Delivering messenger RNA (mRNA) has gained substantial interest for purposes of vaccination due to its multiple advantages. In particular, it has a well-documented safety profile, since it is easily degraded in a biological environment, thus having only a transient expression and avoiding genome integration. It also has the ability to transfect non-dividing, slowly proliferating, and quiescent cells. Moreover, penetration into the nucleus, which is a rate-limiting step, is not required since mRNA translation takes place in the cytosol ,2. In adIn 2021, the world’s first mRNA drug was approved by the US FDA as a vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The concept of an mRNA vaccine was first described in 1993, by Martinon et al., who immunized mice with influenza antigen-encoding mRNA encapsulated in liposomes that were comprised of cholesterol (Chol), dipalmitoyl phosphatidylcholine (DPPC), and phosphatidylserine. They managed to show that, after immunization, re-stimulation of the splenocytes of the mice with virus-infected cells resulted in the development of specific cytotoxic T lymphocyte (CTL) activities. They also demonstrated this response was dependent on mRNA encapsulation and the route of administration of the mRNA-encapsulating liposomes [The lipid nanoparticle system is one of the most promising drug delivery technologies. It is used as the platform for Patisiran, and in vaccines against SARS-CoV-2. Patisiran (ONPATTRO™) is the world’s first lipid nanoparticles siRNA product. It is a therapeutic RNA interference (RNAi) product that is used for the treatment of hereditary transthyretin (TTR)-mediated amyloidosis (hATTR) in adults ,5. PatisLNPs used for systemic administrations of nucleic acids generally contain cholesterol, a pH-sensitive cationic lipid, phospholipids, and polyethylene glycol (PEG). Cholesterol contributes to the stability of LNPs in the blood ,8. PhospThe currently available mRNA vaccines are injected intramuscularly to transfect muscle cells and APCs in the circulation. However, lymphoid organs, such as the lymph nodes and the spleen, contain higher numbers of APCs, which are located near B and T lymphocytes. Therefore, transfecting APCs into lymphoid organs would be expected to produce a more efficient immune response. After the systemic administration of mRNA-loaded nanosystems, most of the dose ends up in the liver. As a result, most of the particles do not reach the intended target organ, and the genetic cargo is unable to produce a therapeutic effect. A specifically designed system that targets and transfect different immune cells in lymphoid organs is needed to reach that goal. For this reason, the main goal of this work was to design mRNA-loaded LNPs that are able to efficiently transfect APCs in the spleen after intravenous (IV) administration, while simultaneously decreasing their accumulation in the liver. The success of this goal would open the possibility of developing novel genetic vaccines for immunotherapy approaches. Such a system could also be applied for the efficient and selective immune stimulation against cancer.It was previously shown that plasmid DNA (pDNA) was successfully delivered to the spleen after IV administration using a lipid system based on the commercially available pH-sensitive lipid 1,2-dioleoyl-3-dimethylammonium propane (DODAP) . DODAP, The optimized composition for pDNA delivery involved the use of a combination of DODAP and DOPE. To test whether DOPE is important or not for gene expression in the case of mRNA delivery, we compared DODAP-LNPs prepared with and without DOPE. The lipid composition and characterization of the different DODAP-LNPs are shown in We attempted to optimize the lipid composition for mRNA so as to improve the activity and selectivity in the spleen. The original lipid composition was similar to that optimized for delivering pDNA to the spleen, i.e., DODAP/DOPE/Chol/DMG-PEG2k, = 18.5/50/30/1.5 mol%. We examined the influence of changing the ratio of DODAP and Chol, the ratio of DOPE and DODAP, and the ratio of total lipid per amount of mRNA. The criterion for the optimal composition was the magnitude of the activity in the spleen and the extent of spleen selectivity. Different LNPs were prepared by the ethanol dilution method and the lipid film hydration method. Since the difference in the level of gene expression produced by LNPs prepared by each method was negligible (data not shown); LNPs prepared by the ethanol dilution method were used for further optimization. The characterization of different LNPs was not significantly altered by changing the lipid composition . We initWe then checked the DOPE/DODAP ratio  with the cholesterol ratio being fixed at 10 mol% of the total lipid C. The lu8 RLU/mg protein. The activity was 41-fold higher in the spleen than that in the liver and 379-fold higher than that in the lung (We then examined the issue of the mRNA-lipid ratio D. The hithe lung E. This iWe compared our optimized LNPs with RNA-lipoplexes (RNA-LPX); one of the strongest known systems for delivering high levels of mRNA to dendritic cells (DCs) of the spleen . The RNAWe next examined the issue of which specific cells in the spleen are internalizing the LNPs. We used specific antibodies against different types of spleen cells for this. To evaluate the cellular uptake in splenocytes, flow cytometry analyses were performed on cells isolated from the spleen. Briefly, 1 mol% DiD-labeled DODAP-LNPs were prepared as described later. These LNPs were then injected intravenously, alongside a phosphate-buffered saline (PBS) control group. After 24 hr, the mice were sacrificed, the spleens isolated, red blood cells were lysed, and the remaining cells were analyzed by flow cytometry. Flow cytometry analyses were performed on four discrete cell populations: B cells (B220+), T cells (CD3+), DCs (CD11c+), and macrophages (F4/80+) . In termWe next evaluated the transfection activity in different cell populations inside the spleen. LNPs encapsulating mRNA-encoding Nluc were injected intravenously at a dose of 0.8 mg/kg, alongside a PBS control group. Four discrete cell populations were sorted by a cell sorter in the same manner as was used for the flow cytometry analysis. Luciferase assays were then performed on these cell populations and the luciferase activity per 3000 cells was calculated C. In theWe successfully developed LNPs that express mRNA to APCs in the spleen in vivo. We tested the potential of the developed mRNA LNPs for use as a prophylaxis against tumors. We evaluated the prophylactic anti-tumor effect of LNPs encapsulating a tumor specific mRNA. In this study, mice were treated with DODAP-LNPs encapsulating the antigen OVA-encoding mRNA (OVA-mRNA LNP), or luciferase-encoding pDNA (Luc-pDNA LNP). Control mice were treated with PBS or naked (OVA)-encoding mRNA (naked OVA-mRNA). Different LNPs and controls were injected at 1 week prior to the inoculation of tumor cells and tumor volume was then monitored . The OVAThe LNP encapsulating OVA-encoding mRNA produced a strong prophylactic anti-tumor effect as shown before. To further examine the issue of whether the IV delivery of LNP induces effector cells that can specifically lyse target cells, we performed an in vivo cytotoxicity assay. At one week before the administration of target and control cells, LNPs encapsulating OVA-encoded mRNA were administered intravenously to immunize the mice, and OVA-specific cytotoxicity was then assessed by flow cytometry. The LNP encapsulating OVA-mRNA (OVA-mRNA LNP) induced strong OVA-specific cytotoxic T lymphocyte (CTL) activities and this activity was dose-dependent A. LNPs eWe also compared our developed LNPs with the RNA-LPX, which showed a high mRNA delivery to DCs of the spleen . The OVAVaccines prepared using an LNP against novel corona viruses are administered by the intramuscular route (IM). It has been reported that the IV administration of the Bacillus Calmette–Guérin (BCG) vaccine, which is generally administered intradermally, increased its efficacy . TherefoWe next examined the therapeutic potential of the developed mRNA vaccine against previously existing tumors. In this study, mice carrying OVA tumors were treated with DODAP-LNPs encapsulating the antigen OVA-encoding mRNA (OVA-mRNA LNP), or luciferase-encoding mRNA (Luc-mRNA LNP) and the RNA-LPX loaded with OVA-encoding mRNA (RNA-LPX). Control mice were treated with PBS or the naked (OVA)-encoding mRNA (naked OVA-mRNA). Different nanoparticles and controls were injected at 8, 11, and 14 days after the inoculation of tumor cells and tumor volume was then monitored . The resDeveloping mRNA vaccines is of great interest and is currently receiving considerable attention for prophylaxis against infectious diseases as well as against tumors. New efficient mRNA-based COVID 19 vaccines are now being approved and are currently being used as the first line of defense against COVID infections. These mRNA vaccines are based on lipid nanoparticles which are injected intramuscularly where they transfect myocytes to produce the spike protein of the virus. They can also transfect APCs that are located near the injection site resulting in antigen presentation through major histocompatibility complex (MHC) proteins which activate antibody production as well as the production of CD8+ T lymphocytes. The transfection of APCs in the spleen or lymph nodes is expected to result in more efficient antigen production, thus leading to a stronger prophylactic effect. However, targeting immune cells in the spleen or lymph nodes is not an easy task and novel gene delivery systems need to be developed for this purpose. The aim of this study was to develop lipid-based nanoparticles encapsulating mRNA which have the ability to transfect immune cells in the spleen after IV administration.We prepared mRNA-loaded lipid nanoparticles in which the main lipid component was the pH-sensitive lipid DODAP and we investigated their transfection activities in different organs after IV administration. We investigated the effect of the inclusion of the helper lipid DOPE on transfection activities in different organs. Since DOPE is a fusogenic lipid, this property contributes to the overall transfection activity by enhancing the endosomal escape . DOPE-coOn the other hand, DODAP is one of the earliest used pH-sensitive cationic lipids used for nucleic acid encapsulation, but the gene delivery efficiency is known to be low ,18. The We performed an optimization study of the lipid composition of mRNA-loaded LNPs in an attempt to obtain the highest gene expression in the spleen. After an examination of the effect of the ratio of DODAP/Chol, we selected a DODAP/Chol ratio of 38.5/10 instead of 48.5/0 because cholesterol is a commonly used ingredient in liposomes and LNPs as a component that stabilizes lipid bilayer by filling in the gaps between phospholipid molecules . Using cAfter optimization, we evaluated the cellular uptake and the luciferase activity in splenocytes. It is well known that macrophages and dendritic cells internalize LNPs by phagocytosis. However, gene expression efficiency is not high in these cells ,22. On tFor purposes of vaccination, the main goal is the transfection of APCs, such as dendritic cells, macrophages, and B cells. These cells will trigger a specific immune response against encoded antigens, thus producing an immune attack against cells that express these antigens. This ends up with their elimination and the reduction in tumor size, in the case of cancer, for example . AlthougOnly a few reports have appeared that confirm the transfection of mRNA in B cells after IV injection, but the transfection efficiency was low ,25. In tIn the prophylactic anti-tumor effect, naked OVA-mRNA inhibited tumor growth to a slight extent . The mRNAlthough the transfection activity in the spleen for the RNA-LPX was higher than that produced from the optimized DODAP-LNP, the CTL activity produced from the RNA-LPX and the DODAP-LNP, at a high dose, was comparable . It shouIn the therapeutic anti-tumor experiment , the DODBoth LNP and RNA-LPX tend to induce a higher CTL activity when administered intravenously than that when administrated intramuscularly . It has DOPE, DOTMA, and DMG-PEG were purchased from the NOF CORPORATION . DODAP and Chol were purchased from Avanti Polar Lipids . Ribogreen assay system was purchased from Thermo Fisher Scientific. Phycoerythrin (PE)-labeled anti-mouse CD3 antibody, PE-labeled anti-mouse CD11c antibody, fluorescein isothiocyanate (FITC)-labeled anti-mouse CD45R/B220 antibody, FITC-labeled anti-mouse F4/80 antibody, purified anti-mouse CD16/32 were purchased from Biolegend . 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD) was purchased from Invitrogen . The luciferase assay reagent and reporter lysis buffer were obtained from Promega . Dulbecco’s modified eagle medium (DMEM), Roswell Park Memorial Institute medium (RPMI), and fetal bovine serum (FBS) were purchased from Sigma-Aldrich Co. . All other materials were of reagent-grade and were commercially available.Four-week-old male ICR mice or female C57BL/6J mice were purchased from Sankyo Labo service . The experimental protocols used in this study were reviewed and approved by the Hokkaido University Animal Care Committee in accordance with the “Guide for the Care and Use of Laboratory Animals”. In all experiments, the animals were used without fasting. Mice were euthanized by cervical dislocation.g, 30 min) with an Amicon tube ultra 100k. LNPs were recovered from the tubes with PBS.The LNPs used in this study were prepared by the ethanol dilution method. An amount of 10 µg mRNA was dissolved in 250 µL 10 mM HEPES buffer (pH 4.0). The lipids were dissolved in ethanol . While stirring the lipid ethanol solution, 250 µL 10 mM HEPES buffer (pH 4.0) containing 10 µg mRNA, 500 µL 10 mM HEPES buffer (pH 4.0) and 1 mL 10 mM HEPES buffer (pH 4.0) × 4 were added in that order. The LNPs solution was centrifuged  and zeta-potential of the LNPs were determined using a Zetasizer Nano . The mRNA encapsulation efficiency and recovery were determined by a Ribogreen assay, LNPs were diluted in 10 mM HEPES buffer pH 7.4 containing 1 mg/mL dextran sulfate and Ribogreen reagent in the presence or absence of 10 w/v% Triton X-100. Fluorescence intensity was measured by INFINITE 200 instrument with λem = 480 nm, λex = 515 nm. mRNA concentrations were calculated from mRNA standard curves. mRNA encapsulation efficiency was calculated by comparing the mRNA concentration in the presence and absence of Triton X-100.DOTMA/DOPE  dissolved in ethanol to a total lipid concentration of 10 mM was added to a glass test tubes and the solvent was removed by an evaporator. After adding 800 µL of D-PBS (-), the contents of the tube were hydrated for about 2 min. The mixture was then agitated and sonicated in a bath sonicator for about 5 min. The prepared liposome solution was diluted 5-fold with D-PBS (-). A 0.173 mg mRNA/mL (28.875 µL) of mRNA solution diluted in D-PBS (-) was added to liposome solution (21.125 µL) with stirring (nitrogen/phosphate (N/P) = 1.3/2). The characterization of RNA-LPX was measured in the same way as LNPs.Four-to-five-week-old ICR mice were treated at a dose of 0.1 or 0.8 mRNA mg/kg body weight (concentration was 26.6 ± 5.6 mRNA µg/mL). The mice were sacrificed at 24 h after the treatment, and the liver, lungs, and spleen were collected. The collected organs were washed with saline, weighed, and minced with scissors. A 0.2 g sample of liver tissue and the other whole collected organs were completely homogenized using a MicroSmash homogenizer in 1 mL of lysis Buffer . After centrifugation at 15,000 rpm for 10 min at 4 °C, a 25 µL aliquot of the supernatant was examined for luciferase activity. The amount of protein in the samples was determined using a BCA protein assay kit (Pierce). Luciferase activities are expressed as relative light units (RLU) per mg of protein.g for 5 min at 4 °C. After removing the supernatant, 1 mL ACK lysis was added and the preparation incubated for 5 min at room temperature. A volume of 9 mL of RPMI medium for the spleen  was added over a period of 5 min at 4 °C. After removing the supernatant, 10 mL RPMI medium was added and washed twice. After cell counting, 1 × 106 cells were blocked with 1.0 µL CD16/32 antibodies in 50 µL FACS buffer and incubated 10 min in ice. The cells were then stained with antibodies for more than 30 min on ice. After incubation, cells were washed with FACS buffer and then cells were sorted via flow cytometry. Luciferase activity was then analyzed.Four-to-five-week-old ICR mice were treated with a dose of 0.8 mRNA mg/kg body weight. The mice were sacrificed at 24 h after the treatment, and the liver, lung, and spleen were collected. The collected organs were then washed with saline, weighed, and minced with scissors. Splenocytes were collected and centrifuged at 500× g, 3 min. The supernatant was removed, the cells were resuspended in medium (10 mL), seeded in 10 cm dishes, and cultured under 37 °C in an atmosphere of 5% CO2 conditions. Passaging was performed every two days. For passaging, cells were collected and then centrifuged at 500× g for 3 min. After the supernatant was removed, the cells were re-suspended in medium and counted. For counting, the cell suspension was mixed with an equal volume of 0.5% trypan blue solution, and only the unstained viable cells were counted.For the E.G-7 OVA cell culture, RPMI 1640 medium containing 50 µM 2-mercaptoethnol, 10 mM HEPES, 1 mM sodium pyruvate, 100 U/mL penicillin-streptomycin, and 10% FBS was used. To start the culture, the cell suspension was quickly lysed at 37 °C, diluted with medium (9 mL), and then centrifuged at 500× 5 antigen (OVA)-expressing E.G7-OVA cells were subcutaneously injected. Tumor volume was calculated by the following formula: (major axis × minor axis2) × 0.52.The mRNA encoding antigen  was purchased from Trilink . In the prophylactic anti-tumor effect experiment, all groups were administered to the C57BL/6 mice by intravenous injection for immunization at 1 week before the tumor inoculation. In the therapeutic anti-tumor effect experiment, all groups were administered to the C57BL/6 mice by intravenous injection at 8, 11, and 14 days after the tumor inoculation. For tumor inoculation, 5 × 106) of CFSE-low (untreated) and CFSE-high (peptide-treated) cells were mixed and intravenously injected into the immunized mice. Splenocytes from the immunized mice were collected 20 h after injection, and single-cell suspensions were analyzed by flow cytometry (CytoFLEX). The numbers of CFSE-low and CFSE-high cells were used to calculate the percentage of peptide-treated cell killing. Peptide-treated cell specific killing was determined as the percentage of specific lysis =  × 100.Antigen specific cytotoxic effect was measured as previously described . C57BL6JIn this study, the goal was to develop LNPs whose activity is selective for the spleen. We succeeded in the selective transfection of mRNA to the spleen after IV administration. The optimized composition of the LNP was found to be DOPE/DODAP/Chol/PEG-DMG2k = 60/28.5/10/1.5 mol%, 240 nmol lipid for 10 µg mRNA. Dendritic cells, macrophages, and B cells in the spleen were all successfully transfected. The developed DODAP-LNPs produced a strong prophylactic effect as well as a therapeutic antitumor effect. Although a lower gene expression was produced in the spleen compared to RNA-LPX, the DODAP-LNPs produced a higher CTL activity and higher antitumor effect. Therefore, we conclude that the developed LNPs are highly promising for use in the treatment of cancer, infections, and other diseases.


Liposomes with adjuvant properties are utilized to carry biomolecules, such as proteins, that are often sensitive to the stressful conditions of liposomal preparation processes. The aim of the present study is to use the aqueous heat method for the preparation of polymer-grafted hybrid liposomes without any additional technique for size reduction. Towards this scope, liposomes were prepared through the combination of two different lipids with adjuvant properties, namely dimethyldioctadecylammonium (DDA) and D-(+)-trehalose 6,6′-dibehenate (TDB) and the amphiphilic block copolymer poly(2-(dimethylamino)ethyl methacrylate)-b-poly(lauryl methacrylate) (PLMA-b-PDMAEMA). For comparison purposes, PAMAM dendrimer generation 4 (PAMAM G4) was also used. Preformulation studies were carried out by differential scanning calorimetry (DSC). The physicochemical characteristics of the prepared hybrid liposomes were evaluated by light scattering and their morphology was evaluated by cryo-TEM. Subsequently, in vitro nanotoxicity studies were performed. Protein-loading studies with bovine serum albumin were carried out to evaluate their encapsulation efficiency. According to the results, PDMAEMA-b-PLMA was successfully incorporated in the lipid bilayer, providing improved physicochemical and morphological characteristics and the ability to carry higher cargos of protein, compared to pure DDA:TDB liposomes, without affecting the biocompatibility profile. In conclusion, the aqueous heat method can be applied in polymer-grafted hybrid liposomes for protein delivery without further size-reduction processes. Dimethyldioctadecylammonium (DDA) is a cationic lipid with several applications in the field of nanomedicines and drug delivery systems ,6,7,8,9.Furthermore, an adjuvant system intended for mucosal vaccination based on biodegradable poly(DL-lactic-co-glycolic acid) (PLGA) nanoparticles modified with the DDA and the immunopotentiator trehalose-6,6′-dibehenate (TDB) lipids was designed and developed by a quality-by-design approach to tailor humoral and cellular immunity characterized by antibodies and Th1/Th17 responses . The particles were separated from the aqueous phase, and the free BSA was analyzed in the supernatant by the bicinchoninic acid (BCA) protein quantification method according to the kit protocol.Protein quantification using BCA  protein assay was carried out following manufacturer’s instructions. Briefly, samples were incubated up to 30 min at 37 °C, with 25 µL of sample and 200 µL of the working reagent. Absorbance was then measured at 562 nm using an INNO microplate reader.supernatantC is the BSA that was quantified in the supernatant (non-entrapped), and totalC is the total concentration of the BSA added in the dispersion [Centrifuged samples of the respective empty nanostructures were also conducted by BCA assay and used as a blank. The loading % was calculated according to the following equation:spersion .m values toward lower temperatures and have been already studied in the literature [m at 51.4 °C and the pre-transition (Ts) at 40.3 °C. These small differences in main and pre-transition temperatures are related to the difference in the dispersion medium. In previous studies, Tris buffer was used, and in this investigation the DSC experiments were run in HPLC-grade water. The presence of salts caused different hydration networks on the surface of lipid bilayers and consequently a slight difference in the Tm values. The presence of PLMA-b-PDMAEMA caused a decrease of the Tm values around 10 °C for all the weight ratios studied. The decrease of Ts was around 7 °C for the 1:0.2:0.5 and 1:0.2:1 weight ratios. At the highest weight ratio of the polymeric guest , the abolishment of the pretransition phenomenon was observed. The enthalpy increased slightly  with the incorporation of polymeric chains into DDA:TDB lipid bilayers (from −117.0 J/mol for DDA:TDB to −113.7 J/mol for DDA:TDB:PLMA-b-PDAMAEMA at a 1:0.2:0.5 weight ratio)  . The inct ratio) . The sigecreased . The decm values , heterogeneous size distribution, and the highest loading efficiency . The sizIn this study, we used the aqueous heat method without size reduction protocol but in low-energy, non-harsh, non-organic solvent conditions for the development of hybrid polymer–lipid delivery platforms with enhanced physicochemical and structural characteristics useful for protein delivery. To the best of our knowledge, this is the first report in the literature where this preparation protocol is used as the formulation process of nanostructures of the type with detailed knowledge of their physicochemical, morphological, thermotropic, nanotoxicity, and protein-loading characteristics.


Despite the absence of Fas-induced cell death treatment of L-MSCs with low concentrations of FasL or anti-Fas mAb resulted in a cellular ATP level decrease, while high concentrations of the inducers caused a decline of the mitochondrial membrane potential. Pre-incubation of L-MSCs with the pro-inflammatory cytokine TNF-α did not promote L-MSC cell death. Our data indicate that human L-MSCs have increased resistance to receptor-mediated cell death even under inflammatory conditions.Mesenchymal stem cells (MSCs) have a pronounced therapeutic potential in various pathological conditions. Though therapeutic effects of MSC transplantation have been studied for a long time, the underlying mechanisms are still not clear. It has been shown that transplanted MSCs are rapidly eliminated, presumably by apoptosis. As the mechanisms of MSC apoptosis are not fully understood, in the present work we analyzed MSC sensitivity to Fas-induced apoptosis using MSCs isolated from the biopsies of liver fibrosis patients (L-MSCs). The level of cell death was analyzed by flow cytometry in the propidium iodide test. The luminescent ATP assay was used to measure cellular ATP levels; and the mitochondrial membrane potential was assessed using the potential-dependent dye JC-1. We found that human L-MSCs were resistant to Fas-induced cell death over a wide range of FasL and anti-Fas mAb concentrations. At the same time, intrinsic death signal inducers CoCl Amor injury . They alr injury . Thus, tr injury ,15.Fas (CD95) is a membrane receptor that belongs to the death receptor superfamily. The CD95 ligand (FasL) belongs to the family of death ligands along with TNF-α and TRAIL. The interaction of FasL with the Fas/CD95 receptor triggers a cascade of signals leading to apoptotic cell death. FasL is expressed either as a membrane-bound protein or in a soluble form . Fas/CD9As repeatedly reported, MSCs can be isolated from various tissue sources and have stable specific characteristics including adhesion to culture plastic, expression of mesenchymal markers CD44, CD90, CD105, and the ability to differentiate into mesodermal lineages. MSC-like cells can also be obtained from the adult human liver . In addi2) were studied. We have shown for the first time that human L-MSCs are resistant to Fas-induced cell death. Neither FasL nor anti-Fas mAb induced cell death in human L-MSCs over a wide range of concentrations. Nevertheless, there was a marked decrease in the level of intracellular ATP after L-MSCs incubation with low doses of FasL or anti-Fas mAb, and mitochondrial depolarization after L-MSC treatment with high doses of FasL or anti-Fas mAb. Pretreatment of the human L-MSCs with the pro-inflammatory cytokine TNF-α did not affect Fas-induced apoptosis resistance of L-MSCs. At the same time, the intrinsic death signal inducers CoCl2 and STS triggered L-MSC cell death in a dose-dependent manner.In the present work, the responses of human L-MSCs to various cell death inducers, such as FasL, anti-Fas mAb, staurosporine (STS), and cobalt (II) chloride (CoClw/v)  and incubated for 30 min in a CO2 incubator. Processed liver tissue was washed twice in PBS  supplemented with 1% FBS . After that, the complete growth medium was added, and the suspension was maintained in culture flasks (75 cm2) until adherent cells appeared. The MSC cultivation was carried out in a complete growth medium DMEM/F12 containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin . The cultivation was carried out under standard conditions in a CO2 incubator . Upon reaching 80–90% confluency, the cells were passaged. In all experiments, the cells from no later than the 5th passage were used.Liver biopsies were provided by the Department of Emergency Surgery and Portal Hypertension of the Petrovsky Russian Research Center of Surgery. The study was approved and supervised by the Institutional Ethics Committee . Written informed consent was obtained from all patients. Biological samples were handled using anonymous codes in accordance with the Federal Law on Personal Data . L-MSCs were isolated from the biopsy material obtained from three patients with liver fibrosis, according to the method described previously . Brieflyg. The cells were incubated with anti-CD14-FITC, anti-CD34-APC, anti-CD45-PE, anti-CD29-APC, anti-CD44-FITC, anti-CD73-PE, anti-CD105-PE, anti-CD95-APC  at concentrations recommended by the manufacturer, for 1 h at 4 °C in the dark. At the end of the incubation, the cells were washed twice in PBS by centrifugation. Fluorescence intensity was analyzed using BD FACSAria III flow cytometer . At least 104 events were recorded in each sample. The results were processed using the FlowJo_V10 . The relative fluorescence intensity (RFI) of marker expression was calculated as the ratio of the specific fluorescence of cell staining with fluorescently labeled antibodies and the autofluorescence of control unstained cells.The cells were trypsinized and washed twice in PBS  supplemented with 1% FBS  by centrifugation for 5 min at 300× 2  at concentrations of 100–400 µM, recombinant human Fas ligand  at concentrations of 50–500 ng/mL and anti-Fas mAb  at concentrations of 0.05–2 µg/mL were used as the cell death inducers. The cells were preliminarily plated in the wells of a 12-well plate (5 × 105 cells/well) until adhesion, after which the inducers were added and L-MSCs were incubated for 72 h.Cell death was assessed by flow cytometry in the propidium iodide (PI) test, as described previously . Stauros4 events were recorded in each sample. The results were processed using the FlowJo_V10 .In order to analyze the effect of pro-inflammatory cytokines on the sensitivity of L-MSCs to Fas-induced cell death, the cells were preincubated with TNF-α (20 ng/mL)  for 48 h and then treated with various concentrations of FasL or anti-Fas mAb  for another 24 h. After incubation, the cells were trypsinized and washed twice in PBS by centrifugation. The cell pellet was resuspended in ice-cold 70% ethanol for 1 h to fix and permeabilize the cells. Then the cells were washed twice in PBS, resuspended in 100 µL PBS-based staining solution supplemented with PI  and RNase  and incubated for 15 min. After this, the volume of the suspension was adjusted to 500 µL with PBS, and fluorescence intensity was analyzed on a BD FACSAria III flow cytometer-sorter . At least 102, or STS  were seeded at 48 h beforehand on a 96-well plate. After that, cell death inducers were added and incubated for 4 h. At the end of incubation, 50 μL of the detergent was added to each well and incubated for 5 min on an orbital shaker at 600–700 rpm. Then, 50 µL of the substrate was added to the wells and incubated for 5 min on an orbital shaker and for an additional 10 min in the dark. Wells with complete growth medium without cells were used as a negative control. Each point in one experiment was run in triplicate. Luminescence was measured on a Tecan infinite M200 Pro plate reader . Data are presented as control-normalized luminescent relative light unit (RLU) values.ATP levels in the L-MSC cultures were determined by the Luminescent ATP Detection Assay Kit  according to the manufacturer’s instructions. The L-MSCs . At least 104 events were recorded in each sample. The results were processed using the FlowJo_V10 .Changes in the level of the mitochondrial membrane potential were analyzed by flow cytometry using the potential-dependent dye JC-1 , as described previously . The L-Mt-test; p < 0.05 was regarded as significant.Statistical data processing was performed using the SigmaPlot 12.5 soft . Data are presented as the mean ± SEM. All measurements were performed in at least three replicates. Statistical differences between groups were calculated by Student’s Human L-MSCs obtained from the liver biopsies of liver fibrosis patients have a fibroblast-like morphology A and areAnalysis of CD95 expression in three cultures of L-MSCs showed that all cell populations express it at a high level B. Probab2 (p > 0.001)  induced through CD95, we also analyzed the intrinsic pathway of cell death induced by STS and CoCl2 . Neither> 0.001) A,B. L-MS> 0.001) . The via> 0.001) . As a pot manner .2 and STS. We have shown a dose-dependent increase in the percentages of dead L-MSCs after treatment with both inducers (p < 0.001).The obtained results may indicate the resistance of human L-MSCs to receptor-mediated cell death via Fas/CD95 ligation. As controls, we also have used two known death inducers: CoClAn important feature of the early stages of the apoptotic cascade is the disruption of the normal functioning of mitochondria, including changes in the membrane potential. Potential-dependent dyes, which freely penetrate the cell plasma membrane, are routinely used to monitor the state of the mitochondria. The JC-1 dye has the property of potential-dependent accumulation in mitochondria. At high mitochondria membrane potential, JC-1 dye monomer forms red fluorescent “J-aggregates” with an emission maximum at ~590 nm (FL2 channel) accumulated within the mitochondria. At a low membrane potential, the JC-1 dye is present as monomers, devoid of the FL2 channel fluorescence and preserving it in the FL1 channel (529 nm). Therefore, a decrease in the intensity ratio of red/green fluorescence indicates depolarization of mitochondria (FL2/FL1) . Using tDespite the fact that anti-Fas mAb or FasL did not induce cell death in L-MSCs in the concentration range studied A,B, shorIt should be noted that only a part of the cells contained depolarized mitochondria after treatment with inducers. 2 was less efficient in the induction of mitochondrial membrane potential in L-MSCs . Close to it, FasL induced a decrease in ATP levels in 60% at 12–25 ng/mL and 30% at the concentration 50 ng/mL (p < 0.001). No effect was found at 100 ng/mL (p > 0.05). High concentrations of anti-Fas mAb (2 µg/mL) or FasL (500 ng/mL), as well as STS and CoCl2, did not lead to the changes in ATP levels in L-MSCs after 4 h incubation  formation to mitochondrial outer membrane permeabilization (MOMP) and subsequent activation of effector caspases and nuclear fragmentation . TherefoMitochondrial depolarization during cell death is associated with the MOMP pathway. MOMP was originally considered an irreversible stage and defined as an all-or-nothing event. However, it has recently been shown that even after MOMP, the mechanisms of cell death regulation can lead to cell survival . EvidentTo date, it has been proven that various cell types become more sensitive to Fas-induced apoptosis when they are in an inflammatory environment. Thus, it is assumed that after transplantation, MSCs, being in inflammatory conditions, become sensitized to cell death signals ,64. SeveOur results indicate that human L-MSCs have an increased resistance to Fas receptor-mediated cell death both in normal and under inflammatory conditions. Whether this fact is a unique feature of L-MSCs or is also characteristic of MSCs obtained from other tissues remains to be established. Due to the lack of knowledge on the L-MSC fate after transplantation into animals, it remains unclear whether apoptosis of these cells occurs in the recipient’s body in vivo. If so, what are the signals and downstream mechanisms that trigger it? Do L-MSCs need any additional stimuli to undergo apoptosis in vivo? And is the potential to form apoptotic bodies in the case of L-MSCs a therapeutically significant process? All these questions remain to be answered in order to make it possible to assess the physiological role and significance of apoptotic cell death for the realization of the therapeutic potential of MSCs.


Our results show that several types of microparticle drug carriers caused hyperproliferation of the tumor when injected IP, as reflected in a reduced survival or an accelerated onset of ascites. Alterations of the product formulation of CaCO3 microparticles could result in less hyperproliferation. The hyperproliferation caused by CaCO3 and PLGA was largely driven by a strong innate immune suppression. A combination with chemotherapy was not able to sufficiently counteract the tumor progression caused by the drug carriers. This research points towards the importance of evaluating a drug carrier before using it in a therapeutic setting, since drug carriers themselves can detrimentally influence tumor progression and immune status of the TME. However, it remains to be determined whether the hyperproliferation in this model will be of relevance in other cancer models or in humans.Novel treatment strategies are needed to provide a better prognosis for ovarian cancer. For this purpose, the current study was designed to evaluate the effects of different types of particle drug carriers on tumor response and on the tumor immune microenvironment (TME) after intraperitoneal (IP) administration in a murine tumor model. Mice with ID8-fLuc ovarian cancer were injected IP with pegylated liposomes, hydroxyapatite, polystyrene, poly(lactic-co-glycolic acid) (PLGA) and calcium carbonate (CaCO Ovarian cancer is the eighth leading cause of cancer-related deaths within the female population worldwide , with hi3) and hydroxyapatite; polymers such as poly (lactic-co-glycolic acid) (PLGA) and polystyrene; and lipid-based, where liposomes are the most well-known example. An important focus in the literature goes towards optimizing intraperitoneal (IP) delivery of chemotherapeutics [3 microparticles were proven to be efficient drug carriers for local delivery of α-radionuclide therapy in preclinical models for ovarian cancer and have also been investigated in models of melanoma [Another type of targeted therapy that is currently investigated is the use of particle drug carriers designed to enhance specific drug retention within the peritoneal cavity in an attempt to increase local efficacy and reduce systemic toxicity of the drug under investigation ,8. A widapeutics ,9. In prapeutics ,11,12,13apeutics ,15. Howeapeutics ,17. Addimelanoma ,19,20.It is clear that drug carriers are of interest in the exploration of new therapeutic avenues. However, less attention is paid to evaluation of their influence on tumor behavior and interaction with the immune system, without the drug of investigation being present. Evidence is emerging that manipulations of the immune system can heavily impact the cancer course ,24,25,266 ID8-fLuc ovarian cancer cells on day 0 of the experiment, in the lower right quadrant of the abdomen. All animal experiments were approved by the ethical committee (P123/2017) and followed the most recent ethical standards  2019/1010) and the ARRIVE  guidelines [The ID8-fLuc cell line was transduced with a lentiviral vector (pCHMWS_CMV-fluc-I-PuroR) by the Laboratory of Molecular Virology and Gene Therapy and Leuven Viral Vector Core in our institute. Female C57BL/6 mice  of seven to nine weeks of age were inoculated intraperitoneally (IP) with 5 × 10idelines ,28.w/v sucrose). The CaCO3 microparticles were prepared as described previously [3-MP-A), 0.9% NaCl and 2.4% (w/w) ethylenediamine tetra(methylene phosphonic acid) (CaCO3-MP-B) or 0.1 M Tris (CaCO3-MP-C). In addition, a non-autoclaved variant in 0.9% NaCl without additive (CaCO3-MP-D) was prepared. Particle diameters were approximately 5, 2, 3.5, 4 and 0.09 µm for CaCO3, polystyrene, PLGA, hydroxyapatite and pegylated liposomes, respectively, as described by the particle manufacturers. All microparticle preparations were made without addition of active drugs. Mass doses for all particle drug carriers ranged between 3 and 10 mg per mouse. All drug carriers were administered through IP injection.Polystyrene , PLGA  and hydroxyapatite  microparticles were dispersed in 0.9% NaCl  to obtain the required concentration. Pegylated liposomes  were used as prepared by the manufacturer  was administered IP at a dose of 1.6 mg/kg. Four out of 30 animals treated with the chemotherapy regimen experienced severe short-term toxicities  as a reaction to the chemotherapy in the first ten days after treatment administration and were sacrificed and excluded from all further data analysis.Carboplatin  was dissolved in Dulbecco’s phosphate-buffered saline  and administered IP at a dose of 80 mg/kg calculated for an average body weight of 20 g per mouse. Pegylated liposomal doxorubicin  gradient, immune cells were isolated from the cell suspension and resuspended in DPBS for flow cytometry.Mice were anesthetized with 50 mg/kg pentobarbital  via an IP injection prior to blood sampling using sodium heparinized microhematocrit capillary tubes. Whole blood was centrifuged at 800 RCF for 10 min. Serum was collected and stored at −80 °C for further cytokine quantification with LuminexDead cells were excluded by the use of eFluor780 fixable viability dye staining . Subsequently, immune cells were stained for extracellular T cell and myeloid markers for which the antibody panels can be found in ®, according to the manufacturers’ protocol using customized ProcartaplexTM Immunoassay Kits . In short, 96-well plates were loaded with antigen-specific capture antibody-coated magnetic beads before samples; standards and blanks were added and incubated for two hours. Subsequently, biotinylated detection antibodies were added. The antibody/antigen complex was visualized using streptavidin-conjugated R-phycoerythrin. A magnetic plate washer was used for washing steps throughout the protocol. Read-out was performed with Bio-plex 200 system of Bio-Rad . Concentrations of the proteins were determined using five parameter log curves generated by the Bio-Plex Manager 4.1.1 software .T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) were measured with LuminexQ = 5%). Data from the immune readouts and bioluminescence imaging were tested for outliers using Grubbs’ method (α = 0.05). Normal distribution was assessed using a Shapiro–Wilk test (n < 8). If data were normally distributed, they were expressed as means and standard deviations; if data were not normally distributed, they were expressed as medians and interquartile ranges. For comparison between all the different groups, an ANOVA with Tukey’s multiple comparisons test or a non-parametric Kruskal–Wallis test with Dunn’s multiple comparisons test was used to compare normally and not normally distributed data, respectively. All data in this manuscript were normally distributed, unless stated otherwise. An (adjusted) p value < 0.05 was considered significant. The power analysis was performed in R version 3.5.1 ; all other statistical analyses were performed using GraphPad Prism version 8.2.1 .A statistical power analysis was performed to determine sample sizes for all experiments. A power of at least 0.80 was reached with 5 to 10 mice per treatment group, depending on the type of experiment. Individual time to ascites development and survival curves were compared using the log-rank (Mantel–Cox) test. Adjustment for multiple comparisons was performed with the Benjamini and Hochberg procedure   compared with vehicle control mice on day 42 post tumor cell inoculation  e. Furtheculation b,c. No o3 microparticles (CaCO3-MP-B) or pegylated liposomes on day 1 post tumor cell inoculation (3 microparticles presented with an increase in M2 macrophages compared with vehicle control mice both at day 28 (padj = 0.0004) and at day 42 post tumor cell inoculation (padj = 0.0095), as well as an increase in the monocytic subtype of MDSCs (mMDSC) at the latter time point (padj = 0.0012) (padj = 0.0212). However, in this case, at the latter analysis time point, an increase in the granulocytic subtype of MDSCs (gMDSC) was observed (padj = 0.0135)  (padj = 0.0158) (3-MP-B (padj < 0.0001), PLGA (padj < 0.0001) and pegylated liposomes (padj = 0.0001)  d,f. Addi 0.0135) d,e. Thes 0.0019) d. Furthe 0.0158) e. A sign 0.0001) g. There padj = 0.0006) and the other particle types  (3-MP-B compared with the control (3-MP-B (padj = 0.0017), which may further corroborate the more favorable immune profile of the liposomes.The differences in immune status between the drug carriers were not translated into any statistically significant effects on survival b, althou 0.0006) c. In con 0.0006) a, the su control b, which  , but the reduction was only statistically significant for the CaCO3-MP-A and CaCO3-MP-D product formulation  . Additionally, for mice that received CaCO3-MP-A, the tumor load measured by bioluminescence imaging at day 28 post tumor cell inoculation was significantly higher compared with vehicle control (padj = 0.0001) and mice that received either CaCO3-MP-C (padj = 0.0015) or CaCO3-MP-D (padj = 0.0001)  b. Intere 0.0001) c.3 microparticle formulations showed an increase in immune suppressive M2 macrophages at the earliest analysis time point (day 14 post tumor cell inoculation) . At the latter analysis time point (day 28 post tumor cell inoculation), the trend of higher fraction of M2 macrophages compared with vehicle control remained but was only significant for the CaCO3-MP-A treatment group (padj = 0.0324), which corresponds to the group of mice that experienced the strongest reduction in survival. The persistent increase in M2 macrophages in this group coincided with an increase in the immune suppressive mMDSC compartment at day 28 compared with the vehicle control mice (padj = 0.0228) (3 microparticle groups over time (3-MP-A group compared with vehicle control (padj = 0.0017), at the latter analysis time point both CaCO3-MP-A and CaCO3-MP-C formulations elicited a significant increase in TIM-3 compared with vehicle control . The increase in innate immune suppression was accompanied with an almost immediate suppression of the cytotoxic CD8 + T cells at day 14 in all mice that received CaCO3 microparticles compared with vehicle control mice , while there was only a trend for the CaCO3-MP-D product formulation (padj = 0.0916). To summarize, most changes in immune status of the tumor were seen between the control and mice that received CaCO3-MP-A, in accordance with this group experiencing the strongest reduction in survival. Although there were trends of changes in some parameters, none were significant compared with the control group in mice receiving CaCO3-MP-D.In accordance with the negative influence on survival, the three CaCOulation) d, althou 0.0228) e. Furthever time f. While rol mice g. Howeve3 microparticle formulations, significant changes were only found when CaCO3-MP-A and CaCO3-MP-D were compared. The fraction of M2 macrophages and concentration of TIM-3 at day 14 was significantly larger in mice who received the CaCO3-MP-A formulation compared with the CaCO3-MP-D formulation (padj = 0.0433 and padj = 0.0078), and similarly, on day 28 the fraction of mMDSCs was higher (padj = 0.0163). These differences were also reflected in the survival  were evaluated (padj = 0.0062).In this experiment, two different dosages (5 mg vs. 2 × 10 mg) of the same CaCOvaluated b. Both g3 and PLGA particles in the ID8-fLuc ovarian cancer mouse model could be counteracted by administering chemotherapy one day after particle administration. When CaCO3-MP-B or PLGA were administered as a single treatment on day 13 post tumor cell inoculation, they both induced a hyperproliferation of the tumor as reflected by a reduced survival compared with vehicle control . The addition of carboplatin and PLD was able to significantly prolong survival compared with single treatment with either CaCO3-MP-B  or PLGA . However, mice injected with CaCO3-MP-B or PLGA in combination with carboplatin and PLD showed a reduced survival compared with mice that were treated with carboplatin and PLD alone . Unfortunately, chemotherapy treatment is only partially able to reverse this hyperprogression  and in different product formulations of the same particle drug carrier (CaCO3), three important remarks have to be highlighted. First, hyperproliferation of the tumor could be minimized by reducing the mass dose of the drug carrier. For the particle drug carriers included in this study, a mass dose of 3 mg was able to eliminate the shortening of survival that is observed with higher doses of 5 or 10 mg. Second, the preparation of the product formulation of the drug carrier is important. For the CaCO3 drug carrier, not all product formulations caused the same degree of hyperproliferation. Moreover, while product formulations CaCO3-MP-A, CaCO3-MP-C and CaCO3-MP-D all caused a shortening of survival in the ID8 model when injected IP on day 1 post tumor cell inoculation, the optimized CaCO3-MP-B product formulation [3-MP-B formulation did not result in a hyperproliferation of the tumor when administered on day 1 post tumor cell inoculation. However, when administered at a later disease stage (day 13), the CaCO3-MP-B did accelerate tumor progression that resulted in a reduced overall survival. This suggests that the tumor reacts differently to the microparticles depending on the tumor burden.The effects from particles on tumor growth has several important implications for preclinical research on IP drug delivery systems for ovarian cancer. Since our results point towards the contribution of innate immune suppression to tumor progression, this highlights the need to use syngeneic mouse models in order to have a fully working immune system within the TME. If only human xenograft models in immunocompromised mice are used in the search for new drug delivery systems, potential immune modulatory effects of the carrier itself may be overlooked. Furthermore, aspects related to product formulation and dosing regimen also require thorough investigation. Although in the current study, hyperproliferation of the tumor is consistently present for different types of particle drug carriers (CaCOmulation  did not We would also like to acknowledge the fact that our study has some limitations. We recognize the lack of detailed information on the activation status and functional properties of the immune cells. However, the focus of our research was more to highlight the general shifts in immune cell populations. Furthermore, we acknowledge that the polarization of macrophages is a broad spectrum of which the pro-tumoral M2-like phenotype is just one extreme . In our To which degree our findings are relevant in a broader scope remains to be seen. Our study focused only on the effects of particle drug carriers in the ID8 mouse model. In order to generalize our findings for ovarian cancer, the results need to be confirmed in other preclinical models. In addition, the current study only assessed whether chemotherapy treatment could reverse the tumor hyperproliferation caused by microparticles when added to the schedule as a separate treatment. To properly assess whether the pro-tumoral effects of the carrier itself can be counteracted, chemotherapy-loaded microparticles would have to be compared with standard chemotherapy treatment in order to take advantage of the prolonged IP residence time of the drug. This, along with systematic studies of the interactions between the immune system and drug carriers, can potentially contribute to an improved design of IP drug delivery systems in the future.Intraperitoneal drug delivery by use of particle carriers is a potential new therapeutic option for ovarian cancer patients. Herein we provide essential information on the effects of several commonly investigated drug carriers on disease progression and TME in the ID8-fLuc mouse model, when the particles were administered without a drug payload. With this research we want to create awareness of the possible detrimental effects of drug carriers themselves, as we demonstrated that an IP injection of particles can result in hyperproliferation of the tumor, mediated by strong innate immune suppression. The results emphasize the need for using syngeneic tumor models when developing drug delivery systems and the importance of product formulation and dosing regimen, as reduction in the mass dose, changes in the formulation and adjustment of the timing of injection could diminish the negative effects. Therefore, we believe that these findings can contribute to future developments of novel and better targeted therapeutic strategies for ovarian cancer.


Drug administration to the wound site is a potential method for wound healing. The drug retention duration should be extended, and drug permeability through the buccal mucosal layer should be regulated. Oral wounds can be caused by inflammation, ulcers, trauma, or pathological lesions; if these wounds are not treated properly, they can lead to pain, infection, and subsequent undesirable scarring. This study aimed to develop Kolliphor-407 P-based gel containing neomycin sulfate (NES) loaded in solid lipid nanoparticles (SLNs) and enhance the antimicrobial activity. By considering lipid concentrations and achieving the lowest particle size (Y1) and maximum entrapment (EE-Y2) effectiveness, the formulation of NES-SLN was optimized using the Box–Behnken design. For the selected responses, 17 runs were formulated (as anticipated by the Design-Expert software) and evaluated accordingly. The optimized formulation could achieve a particle size of 196.25 and EE of 89.27% and was further utilized to prepare the gel formulation. The NES-SLN-G formula was discovered to have a smooth, homogeneous structure and good mechanical and rheological properties. After 24 h of treatment, NES-SLN-G showed a regulated in vitro drug release pattern, excellent ex vivo permeability, and increased in vitro antibacterial activity. These findings indicate the potential application of NES-SLN-loaded gels as a promising formulation for buccal mucosal wound healing. Wound healing is a dynamic and complex process involving the replacement of devitalized cellular structures and tissue layers. A successful wound dressing should keep the site moist, allow for gas exchange, act as a microbial barrier, and eliminate excess exudates . Oral woHydrogel is a three-dimensional 3D) network made up of hydrophilic polymer chains that can hold abundant water in their structures, allowing it to be used as a drug carrier system D network,5. VarioSolid lipid nanoparticles (SLNs) are colloidal lipid nanocarriers with a solid core. They are one of the ideal medication delivery devices since they are made of biocompatible materials . SLNs caFurthermore, SLNs are an effective dermal drug delivery method, with improved dermal penetration, occlusive properties, and a longer residence duration inside the skin layers . The intPharmaceutical development is a lengthy and complex process that begins with the formulation and ends with the finished product. In any case, the pharmaceutical product should provide the patient with a certain level of quality. Despite advances in pharmaceutical technology and medicine manufacturing, the pharmaceutical industry still relies heavily on traditional process development and drug manufacturing methods. The classic formulation development technique employs raw material controls, product manufacturing controls, in-process controls, and final product controls to ensure product quality.As a result, detecting the link between product features and product quality using an empirical technique is challenging, and quality is not always guaranteed . Interna−1 were due to NH stretching, OH stretching, and C-H stretching (aromatic), respectively. C=C stretching and C-H stretching were observed at 1521.57 and 3898.75 cm−1, and 1628.52 and 1119.43 cm−1 correspond to C-O and C=O stretching. In the end, these bands were identified in the formula. The spectrum revealed that pure NES and selected ingredients were mutually compatible. The characteristic band of NES and the physical mixture of optimized formulation ingredients are shown in The optimal degree of the parameters chosen and their interconnection in achieving desirable EE and mucoadhesion qualities was evaluated using the Box–Behnken design of response surface methodology (RSM). The quadratic model was chosen for all responses based on the sum of squares (Type I) and fit summary (adjusted and projected R2) . A quadrp-values for both responses were insignificant  and density [0.996 ± 0.05 and 0.998 ± 0.03] of the NES-SLN-G and NES-G gel formulations were similar to those of deionized water.The NES-SLN-based gel had a smooth, uniform composition with a pH of 5.85 ± 0.15, making it appropriate for topical use. The final gel formulation’s viscosity was determined to be 92,105 mPa∙s. Similarly, the value of spreadability was determined to be 5.92 g.cm/sec, indicating that the gel could be spread easily with minimal shear. Even with a blank gel, the same findings were obtained. Surface tension governs the interactions between two immiscible substances. With increasing surfactant concentration, the surface tension of the SLNs usually decreases. The higher surfactant content reduces the surface area of each particle in the formulation by reducing the superficial tension. The presence of optimized surfactant concentration with additional surfactant properties of glyceryl monostearate was found to have a minimum surface tension of 20.52 ± 1.52 dynes/cmNES release, a valuable indicator of in vivo drug performance, was examined using a modified Franz diffusion cell at a skin temperature of 32+− 2 °C. The Franz diffusion cell had a cellulose acetate membrane with a pore size of 0.45 m clamped between the donor and recipient compartments. The drug release from the SLNs was biphasic, with a burst of drug release followed by regulated drug release . The druS. aureus and E. coli. After 24 h of treatment, the Kolliphor-407 P gel containing NES-SLN had more significant antibacterial activity against S. aureus than the pure NES gel (p < 0.05)  . Recent This investigation aimed to see how far the prepared NES-SLN-G could penetrate the skin. These findings imply that compounded SLN-G has the potential to improve medication bioavailability by improving NES mucosal membrane penetration deep into the dermal region. Furthermore, because no discrete pores were detected, the permeation was thought to be transcellular. Because the produced particles are lipid-based, their penetration favors direct passage through the phospholipid-based cell membrane.The stability of the gel formulation, including MTZ SLNs, was assessed by measuring particle size and in vitro drug release from HEC gels maintained at room temperature  for one month. After 1 month of storage, the particle size of NES-SLN in the gel formulation remained stable [197.3 nm] compared to the initial content [196.5 nm]. As evidenced by the f2 value of 82.54, the release profiles of the gel formulations followed the same trends over time (which is more than 50). The inclusion of NES-SLN in the gel formulation helped its stability over a one-month storage period, according to the stability study results.NES was purchased from India-based Yarrow Chemicals, Mumbai. Sigma-Aldrich Co. provided stearic acid, Pluronic F 68, and Glycerol monostearate . All additional compounds and solvents used were of analytical quality and were not purified further.To look for important interactions, pure drug and excipients were analyzed using an FTIR spectrometer  using the KBr pellet method in a range between 4000–450 cm-1 to identify the interactivity ,30.w/v) and NES (0.5%), were dissolved. This solution was injected at a rate of 1 mL/min using a 0.45 mm/26 G  needle into 100 mL of aqueous phase (purified water) containing 0.25 g of non-ionic surfactant Pluronic F 68 [P-F68] at room temperature (25 °C) under continuous stirring (2000 rpm for 2 h). After that, the solvent was evaporated for 5 h in a vacuum oven at 40 °C and 200–400 mbar.The modified solvent injection approach  was usedTo investigate the interaction of independent variables, the Box–Behnken design was chosen. The percentages of Stearic acid A (X1), Glycerol monostearate (X2), and P-F 68 were chosen as the three factors (X3). These variables’ effects on particle size (Y1) and entrapment efficacy (Y2) were investigated. Photon correlation spectroscopy (PCS) was used to measure the particle size (Z-average mean) of prepared SLNs, and each measurement was performed in triplicate using a Zetasizer .T = total amount of NES used in the preparation of nanoparticles, and RPF = free NES present in the supernatantEE is the ratio of the absolute theoretical amount of drug entrapped in the polymer to the practical amount entrapped in the polymer ,33,34. FThe responses generated by all of the preparations were triggered using Design-Expert software. The responses were used to create the methodology for the study and the response surface graph. A numerical standardization method was used to build an upgraded preparation with a stated minimal and maximal limit for each parameter. The findings were combined to create a desirability function ,36. The w/w) was used as a gelling agent to formulate NES-SLN-based gel after optimizing the selected variables [w/w Kolliphor-407 P dispersion for comparative study (NES-G).Kolliphor-407 P , surface charge, morphology, density, surface tension, viscosity, pH, and spreadability to anticipate their appropriateness for topical application.At 25 °C, the particle size and particle size distribution were investigated using a Zetasizer . In brief, 10 L of the material was mixed well with 1 L of deionized water and vortex-mixed for 2 min, and photon correlation spectroscopic analysis was performed as previously described . The aveA Zetasizer was used to assess the surface charge of manufactured SLN colloidal dispersions . As previously stated, an aliquot of 700 L of non-diluted colloidal dispersion was supplied to the Zeta potential cell, and its charge was analyzed in triplicate.SEM was conducted to study the morphology features of the optimized microsphere formulation  . The samAt 25 ± 1 °C, the density of produced SLNs was measured using an ordinary pycnometer. The weight of the empty pycnometer of known volume (V) was first recorded as W1, and the weight of the water-filled pycnometer was recorded as W2 (W2). W3 was the weight of the pycnometer loaded with SLNs. The following equation (5) was used to calculate the density of SLNs .Densityγ1: Surface tension of dispersion;γ2: Surface tension of water;δ1: Density of dispersion;δ2: Density of water;n1: Number of drops of SLNs;n2: Number of drops of deionized water.The surface tension of the SLN dispersions was measured using a laboratory stalagmometer. The instrument was cleaned with deionized water before being filled with double-distilled water up to point A. The full water was then run down to point B, drop by drop, while the number of drops was counted. For the SLN dispersions, the same methods were followed. As noted in the previous section, the density of SLNs had already been computed. Following that, the surface tension of SLNs was computed using Equation (7) .γ1 =  with a Helipath stand was used to measure the viscosity of the NES-SLN-G and NES-G formulations. The sample was placed in a beaker and allowed to equilibrate for 5 min before using a T-C spindle at 0.5, 1, 2.5, and 5 rpm to measure the dial reading. At room temperature, the measurements were repeated in duplicate .S = Spreadability of gel,M = Weight (g) applied on the upper plate,L = Length (cm) of the glass plates,T = Time taken for plates to slide the entire lengthA digital pH meter was used to determine the pH of the prepared gels . Spreadability (S) was determined by placing 2.5 g over one of the slides, which was then covered with a second glass plate. For 5 min, a weight of 1000 g was permitted to rest on the upper glass plate. After removing the weight, the distance traveled by the upper slide was computed, and spreadability was estimated using the formula.Drug release from the developed SLNs and their related gels was determined using a Franz diffusion cell. A 0.45 m cellulose acetate membrane was attached between the donor and receptor compartments ,43. To iStaphylococcus aureus ATCC 29,213 and Escherichia coli ATCC 25922. Fresh cultures of both bacteria were injected onto the plates at an optical density of 108 bacteria/mL  in a sterile 0.9 percent salt solution. Each well (6 mm in diameter) was made by removing the agar with the back of a sterile Pasteur pipette after drying at room temperature for 15 min. Each well received the produced gel formulations containing NES or NES SLNs (0.1 g). The plates were then incubated for 24 h at 37 degrees Celsius. Each assay was carried out three times. The inhibition zones were measured with a 0.1 mm precision caliper . As a control, the Kolliphor-407 P gel formulation without NES was employed [The antibacterial activity of the produced gels was tested using Mueller–Hinton agar plates and the plate diffusion method. Gram-positive and Gram-negative bacteria were employed, namely, employed .The drug was substituted with rhodamine at a 0.15 mol/mL concentration in NES-SLN-G, as described before. Rhodamine is a group of related dyes that are a subset of the triarylmethane dyes and are xanthene derivatives . DialysiThe physical and chemical stability of the Kolliphor-407 P gel containing NES SLNs was assessed by quantifying the particle size of NES SLNs in the Kolliphor-407 P gel, as well as the in vitro drug release over a month of storage at room temperature  .This research looked at the adaptability of QbD in the lab and provided high-level suggestions on the scope and applicability of QbD. The research was carried out to formulate solid lipid nanoparticles, and the integration of the QbD concept with modern methodologies was confirmed to be a significant advancement for the pharmaceutical industry since this time- and cost-saving process ensures a high-quality result. The Box–Behnken design was used to successfully optimize NES-SLNs. According to the desirability approach, a formulation having 0.467 percent stearic acid, 0.275 percent glyceryl monostearate, and 1.23 percent P-F 68 can achieve the optimal formulation parameters for NES-SLN preparation. The relative inaccuracy was found to be within acceptable bounds, validating the design’s correctness. Particle size, PDI, and zeta potential were assessed after the improved formulation was successfully manufactured in gel systems. SEM analysis indicated that the lipid nanoparticles did not aggregate and that the formulation was dispersed uniformly. In comparison to NES-G and NES-SLN, drug release experiments showed that the NES-SLN-G formulation had a more regulated release. Owing to the enhanced antibacterial activity, the designed gel formulation was proven to be efficacious in treating wounds.


ZA-8 exhibited the highest fill factor and efficiency of 0.37% and 0.69%, respectively, because of its boosted short-circuit current and open-circuit voltage.This study reports a simple new technique for the preparation of novel hexagonal-shaped mixed metal oxides (MMO) nanorods using Zn/Al-layered double hydroxide (LDH) as a precursor for dye-sensitized solar cell (DSSC) application. The effect of the Zn to Al molar ratio demonstrated a sound correlation between the obtained nanorods’ diameter and the fabricated DSSCs efficiency. Additionally, the optical behavior of the fabricated MMO film as well as the absorption enhancement due to the utilized dye are also demonstrated; a cut-off phenomenon at around 376 nm corresponds to the attained hexagonal nanorods. The open-circuit voltage augmented noticeably from 0.6 to 0.64 V alongside an increase in the diameter of nanorods from 64 to 80 nm. The results indicated that an increment in the diameter of the nanorods is desirable due to the enhanced surface area through which a higher amount of dye N719 was loaded ( Howevications ,16,17,182@MMO” with plate-like morphology; a conversion efficiency of 1.5% was attained due to the active role of TiO2 in the MMO matrix [A number of reports in the literature report the utilization of MMOs-based Zn/Al-LDHs nanostructures for DSSC applications. Zhang et al. demonstrated the occurrence of hexagonal plate-like MMO particles through which the highest efficiency obtained was 0.0129% . The samO matrix . AdditioO matrix . HexagonO matrix . Wang etO matrix . DespiteIn this attempt, this study reports a simple technique for the preparation of novel hexagonal-shaped MMO nanorods using Zn/Al-LDH as a precursor. The obtained MMO nanorod layers were used for the fabrication of photo-electrode for DSSC application. Subsequently, a robust correlation between the diameter of the nanorods, Zn to Al molar ratio, and dye loading amount was established.2+ to Al3+ (m), whereby x M of Zn(NO3)2·6H2O and y M of Al(NO3)3·9H2O under an ambient environment and a constant stirring rate of 750 rpm. Essentially, the pH of the growth solution was preserved at 7 throughout the experiment  for the conservancy of a homogeneous growth matrix. Subsequently, the obtained white precipitate was processed in an autoclave at 65 °C for 8 h to confirm a successful structure growth. Herein, the subsequent solution was later spin-coated on fluorine-doped tin oxide  substrate to form a 1 cm2 layer. The deposition process was repeated for three cycles in which each cycle was subject to an annealing process at 90 °C for 30 min. Phase transformation of the fabricated layers from LDH to MMO was obtained via a thermal treatment process at 350 °C for 1 h with a heating rate of 5 °C/min. Finally, the fabricated layers were denoted as ZA-m, where m designates the molar ratio.Hexagonal-shaped Zn(Al)O-MMO nanorods derived from Zn/Al-LDH were synthesized via a combination of co-precipitation and hydrothermal methods. In particular, Zn/Al-LDH precursor was synthesized as a function of the molar ratio of ZnZA-m layers were immersed in a dye solution containing 5 mM of Ruthenizer 535-bisTBA  for 3 h; the resultant layers were used as the photo-electrode. Afterwards, both the photo-electrode and the counter-electrode were assembled together using an adhesive polymer film , which acts as a sealing and separator element. Hereinafter, electrolyte Iodolyte Z50 redox couple: iodide/triiodide (Solaronix) was fully captivated onto the fabricated electrodes via capillarity.In this study, the Pt electrode (50 nm) was deposited on the FTO substrate using a sputtering technique on the counter electrode. Concurrently, the fabricated −2 under simulated AM 1.5 G sunlight.The thermal stability of the Zn/Al-LDH precursor was investigated via thermogravimetric and differential thermal analysis  with a heating rate of 5 °C/min. The structural and morphological features of the fabricated layers were, respectively, studied using X-ray diffraction  under CuKα radiation and 40 kV, as well as field emission scanning electron microscopy  operated using 1.20 kV accelerating voltage in conjunction with energy dispersive X-ray analysis (EDX). The optical analysis of the fabricated layers was evaluated using UV–vis spectrometry . Finally, the photovoltaic behavior was performed using Keithley 237 SMU  with an output intensity of 100 mW/cmZA-LDH dehydration, and ZnO recrystallization as well as carbonate decomposition.2 structure; this was found to be in accordance with (JCPDS-No:38-0486). Subsequently, the 2D layers structure of LDH collapse, as well as the ZnO crystal formation, can be seen in 3+ within the MMO matrix [3+ in the MMO matrix was confirmed using the EDX technique , while samples ZA-7 and ZA-8 revealed relatively longer hexagonal-nanorods diameters of 75 and 80 nm, respectively. Hence, it is equitable to suggest that there is a direct correlation between the nanorods crystal variation of the deposited MMO and the molar ratio used in this study. The FESEM images also indicate the occurrence of a nanopencil-like morphology, inset in 3+ within the MMO matrix was confirmed through EDX analysis The top-prospective of the deposited hexagonal ZA-8), shifting of absorption edge towards a higher wavelength, the so-called red-shift. Continuously, the absorption spectrum of ZA-8 together with ZA-8 containing N79 sensitizer is demonstrated in ZA-m nanorods evidencing that a larger nanorod diameter leads to advanced N719 loading (The absorbance spectra of the deposited MMO layers are elucidated in ablished d. Partic loading d. Such aThe energy band diagram of the deposited MMO layers and the utilized N719 are presented in ZA-8 exhibited the highest ZA-6 and ZA-7). DSSCs fabricated based on ZA-6 and ZA-7 showed an efficiency of 0.41% and 0.59%, respectively, showing a 20% efficiency enhancement. The demonstrated outcomes agree well with the discussed analysis (FESEM and estimation of dye loading) that a high molar ratio delivers high DSSC performance ; however, an efficiency of 1.02% was reported by Xu et al. using the same materials. It should be mentioned that the aforementioned research group proposed a different structure, sheet-like hexagonal MMO particles. Furthermore, Foruzin et al. revealed the occurrence of efficiency as high as 1.5%, which is mainly due to the active role of TiO2 within the MMO matrix.Continuously, the current density  and efficiency (0.69%), which can be attributed to the enhanced short-circuit current and open-circuit voltage.An easy and new approach for the synthesis of hexagonal MMO nanorods utilizing LDH as a precursor was successfully proposed for DSSC application. The deposited MMO films were characterized using TGA/DTG, XRD, FESEM, and UV-Vis techniques. In particular, the UV-Vis analysis revealed a cut-off phenomenon at 376 nm, which corresponds to the attained hexagonal MMO nanorods. Herein, the effect of the molar ratio between the employed metals (Zn and Al) delivered a pronounced correlation between the microstructural and electrical characteristics of the prepared MMO nanorods and the fabricated DSSCs, respectively. Specifically, increasing the attained MMO nanorods diameter from 64 to 80 nm resulted in higher open-circuit voltage, from 0.6 to 0.64 V, respectively. This was particularly crucial as a larger diameter demonstrated a higher utilized dye N719 loading onto the deposited MMO layers (